Mittelbaukurse June 18-July 5,...

Plant Developmental Biology – TUM: Lab Course

Lab Course 2011Plant Developmental Biology

TU München

Instructors

Prof Dr Kay SchneitzBalaji Enugutti

Charlotte KirchhelleChristine SkorniaPrasad Vaddepalli

- 1 -


Table of contents

General Information

1. Literature Discussion 3

2. Presentation 3

3. Lab Protocols 3

4. Participants 3

5. Lectures 3

6. Schedule 4

Course Schedule

Lab 1: Plant Anatomy and Development 7

Lab 2: CSLM Analysis of GFP Localisation in Ath. Roots. 13

Lab 3: Clonal Analysis and Fate Mapping 20

Lab 4: Genetic Analysis of Flower Development 26

Lab 5: Activation Tagging and Gene Isolation 31

Lab 6: Analysis of Gene, Literature Search, Presentation of Results 50

- 2 -


General Information

1. Literature discussion

The teaching assistants (TAs) will assist you in finding the appropriate literature you will present at the end of the course.

2. Presentation

Students will prepare a power point presentation to discuss some of their results at the end of the course. Presentation will be in English.

3. Lab protocols

Written experimental protocols (English) should be delivered to the secretary at the end

of the course.

The FINAL DATE is Monday 14th of November 2011!!

4. Participants

5. LecturesTable 1

Lecture Date SpeakerIntroduction October 11th Kay Schneitz

Fate mapping/clonal analysis October 12th Charlotte Kirchhelle

Flowers/homeotic genes October 13th Kay Schneitz

Plant transformation/reporter genes October 13th Prasad Vaddepalli

Activation tagging October 18th Balaji Enugutti

Sequencing/BLAST search October 19th Christine Skornia

Demonstration: Literature search October 24th Christine/Balaji

- 3 -


6. Schedule

TimeTuesday

October 11th

Wednesday

October 12th

Thursday

October 13th

9:00 - 10:00 Lecture: Introduction

Lecture: Clonal

analysis, fate

mapping

Lecture: Flowers,

homeotic genes

10:00 - 11:00Plant Anatomy and

Development

Clonal analysis, fate

mappingFloral homeotics

11:00 - 12:00 Ac::GUS Floral homeotics

Lunch

13:00 - 14:00CLSM imaging of

GFP in Arabidopsis

Lecture: plant

transformation,

reporter genes

14:00 - 15:00 HOM::GUS

15:00 - 16:00Results and

Discussion

16:00 - 17:00

Results and

Discussion

- 4 -


TimeTuesday

October 18th Wednesday

October 19thThursday

October 20thFriday January October 21st

9:00 - 10:00Lecture: Activation

Tagging (AT)

AT: Secondary TAIL-

PCR

AT: prepare gels

For product

purification

Time to write your

protocol

10:00 - 11:00 AT: look at plants

Lecture:

Sequencing/Sequence

Comparisons (BLAST)

AT: Product

purification

OR

Repeat any

experiment required

11:00 - 12:00

Lunch

13:00 - 14:00 AT: extract DNAAT:Send products for

sequencing.

14:00 - 15:00 AT: measure DNA

AT: Set up Tertiary

TAIL-PCR and

sequencing reaction

15:00 - 16:00AT: Set up

Primary TAIL-PCR

16:00 - 17:00

- 5 -


Time MondayOctober 24th

TuesdayOctober 25th

WednesdayOctober 26th

ThursdayOctober 27th

9:00 - 10:00AT: get back

sequences Literature searchPreparation of

presentations

Preparation of

presentations

10:00 - 11:00 AT: BLAST

analysis

11:00 – 12:00 Read papers

Lunch

13:00 - 14:00Demonstration:

Literature SearchPreparation of

presentations Student

Presentations (E)14:00 - 15:00

Literature search

15:00 - 16:00 Cheese and Wine

16:00 - 17:00

- 6 -


TUM Plant Developmental Biology Course October 2011

Lab 1: Plant Anatomy and Development

- 7 -


IntroductionThis laboratory will introduce you to the structure of wild-type Arabidopsis plants

at both the whole plant morphology and internal anatomy levels. We will

emphasize the organization and identification of cell and tissue types within the

vegetative part of the plant body, the stems, leaves and roots. We will also

discuss the relationship between cell structure and function and the

developmental relationships between the various cell and tissue types and their

progenitors in the embryo and apical meristems. Our primary goal is to gain

experience in the interpretation of the mature structure of wild-type Arabidopsis

plants (and the developmental basis for that structure) as a baseline for future

comparison with mutant phenotypes.

In this lab we will use several methods that allow rapid microscopic

examination of plant cells and tissues with minimum distortion: clearings,

surface impressions, epidermal peels and toluidine blue O-stained hand

sections of living tissue. Much can be learned about the plant phenotype from

these methods, and these are a good place to start before embarking on the

more time-consuming protocols for preparation of tissues for light, electron and

confocal microscopy.

Plant morphology

1. Obtain a plate of wild-type Arabidopsis seedlings. Gently pull the

seedlings from the agar and mount on a microscope slide in lactophenol.

Coverslip and observe using a compound microscope.

Identify cotyledons, hypocotyl, and primary root bearing emerging lateral

roots. Locate the shoot apical meristem region surrounded by developing

leaf primordia and root apical meristem protected by a root cap. Observe

vascular tissue, noting the continuity of the vasculature throughout the

plant.

2. Examine the shoot of older Arabidopsis seedlings growing on soil. Note

the helical phyllotaxis and leaf heteroblasty. Starting with the cotyledons,

- 8 -


remove leaves one at a time in the order that they were formed, and

mount on double stick tape. How do successively formed leaves differ in

size, shape and trichome distribution? How many juvenile leaves and

how many adult leaves are produced?

3. Examine Arabidopsis plants that are flowering. Once the stem has

elongated, it is possible to recognize the reiterated basic units of shoot

construction, the metamers (each consisting of leaf, axillary bud and

internode). Compare type 1, type 2, and type 3 metamers (Schultz and

Haughn, 1991).

Surface micromorphology

1. Pattern, shape and size of surface cells can be determined quickly by

isolating the epidermal layer or by making a surface impression. First,

practice removing the abaxial epidermal layer from a Kalenchoe leaf by

making two shallow cuts in the margin, grasping the tab of leaf tissue

with forceps, and pulling toward the midvein. Mount the epidermal strip in

water on a microscope slide, coverslip, and observe. Identify epidermal

cells, guard cells and trichomes.

2. Making an epidermal peel is not as easy with Arabidopsis! Two other

approaches that can be used are: (1) Make a surface impression using

cellulose nitrate (a.k.a. clear nail polish). Brush on a thin layer, let dry two

minutes, use forceps to pull off, and mount dry on a microscope slide.

Coverslip and examine. (2) Isolate a living epidermal layer using medical

adhesive spray. Spray medical adhesive spray on a glass microscope

slide and spread using a Kimwipe. Let the adhesive dry for 5 min and

then press the upper surface of an Arabidopsis leaf gently onto the

spray. Flood the upper surface of the leaf with water and then gently

scrape away the lower epidermis, mesophyll and veins, using a single

edge razor blade. Mount coverslip in a drop of water and examine.

- 9 -


3. Compare your observations with scanning electron micrographs of

surface replicas of intact Arabidopsis leaves.

Internal anatomy

1. Start with the large stems and easy-to-identify phloem of a squash

(Cucurbita) stem. Remove a 1-2 centimeter section of stem and cut cross

sections using a double edge razor blade. Stain for 30 sec. in 0.05%

aqueous toluidine blue O (TBO) in a watch glass (or petri dish), rinse in

water in a second watch glass, and transfer to a drop of water on a

microscope slide. Coverslip and observe. TBO is a metachromatic dye

that stains lignified secondary cells walls blue/green and pectin-rich cell

walls purple/pink.

Locate the three tissue systems and their component tissues. Note that

the vascular bundles of squash stem are unusual in having both internal

and external phloem.

Cut longitudinal sections, stain with 0.05% TBO and examine. Identify

xylem tracheary elements, phloem sieve tubes, parenchyma,

collenchyma, sclerenchyma, and epidermal cells.

Aniline blue fluorescence can be used to identify phloem sieve tubes.

Mount unstained sections in 0.1% aniline blue and observe using a

fluorescence microscope.

2. Section, stain and observe a wild-type Arabidopsis inflorescence stem.

Note dermal tissue with epidermal cells, stomates and trichomes,

vascular tissue with xylem and phloem, and ground tissue with

chlorenchyma, sclerenchyma and parenchyma cells. Small or thin plant

parts can be more readily sectioned if supported between pieces of

carrot (make a slit about halfway down a 3 cm carrot stick; insert the

plant material to be sectioned, hold firmly, wet razor blade and plant

material in a petri dish of water, and hold horizontally to section.

3. Use the carrot method to make cross sections of Arabidopsis leaves.

Identify epidermal layers, palisade and spongy mesophyll, and veins.

- 10 -


4. Compare your freehand sections with prepared slides of Arabidopsis

stem, leaf and root cross sections (shown by the TAs). The tissue on

these slides has been chemically fixed, dehydrated, and embedded in

Spurr's resin. The sections reveal detailed structure because they are

thin (2m thick), but tissue also has been altered by the fixation,

dehydration, and sectioning processes.

Locate the Arabidopsis root cross sections on the prepared slides (next

to black line). Identify dermal, cortex, and vascular tissue (stele). Locate

the endodermal layer and xylem and phloem of the stele. Compare the

appearance of these layers in cross section with the GUS-stained

cleared seedling roots.

- 11 -


Analysis of locations of cell cycling using a GUS reporter construct

1. Obtain cyc1At::GUS Arabidopsis seedlings. They have been stained

previously by the TAs.

2. Mount the seedlings in lactophenol on a microscope slide, and coverslip.

First observe the seedlings under a dissecting microscope and then

under a compound microscope. Identify GUS-stained cells. Determine

the relative frequency of cells with GUS activity in the root apical

meristem, sites of initiation of lateral roots, the shoot apex region, and

expanding leaves.

3. Repeat with older plant material, i.e. rosette leaf stage and after bolting.

Focus on above-ground tissue. Compare main SAM and lateral

meristems. Look at IM, flower primordia and floral organ primordia.

Compare floral organ primordia of various stages.

- 12 -



Lab 2:

CLSM Analysis of GFP Localisation

In Arabidopsis Roots

- 13 -


Visualisation of the Arabidopsis root apex and of GFP-labelled subcellular structures in Arabidopsis cells using confocal laser scanning microscope (CLSM)

Introduction

Confocal laser scanning microscopy (CLSM) represents one of the most

significant advances in optical microscopy ever developed. This technique

enables visualization deep within both living and fixed cells and tissues and

affords the ability to collect sharply defined images of cellular components or of

cells as a whole.

A fundamental aspect of confocal microscopy is the use of fluorescent

molecules. Fluorescent dyes and fluorescent protein tags, such as GFP, are

used to highlight known structures within cells. When excited with light, these

molecules emit light at a lower wavelength that can be detected as an image.

As a result, the labeled cellular components are visualized. The microscope

itself scans precise focal planes to obtain optical sections of a specimen, that is,

a 2-dimensional image of that specimen at a particular plane. When a series of

these sections (a Z-stack) is obtained, it can be rendered into a 3-dimensional

image of that specimen. Thus, confocal microscopy, in conjunction with

fluorescent labels, can provide insights into three-dimensional cell and tissue

morphology in organisms, as well as subcellular structures within cells.

Importantly, confocal microscopy enables live cell imaging, where dynamic

processes can be observed such as cell division, chromosome replication and

organelle dynamics, or the activities of a particular protein of interest.

In this lab we will demonstrate two common applications of CLSM using

Arabidopsis as a model: the three-dimensional visualization of cellular

organization in the root apex and the imaging of GFP-tagged subcellular

structures within cells. Our imaging instrument is the Olympus FluoView 1000

with an IX81 inverted microscope stand. The primary aim is to gain an

- 14 -

http://www.olympus-europa.com/medical/22_FV1000.htm


appreciation of how cellular structures can be imaged through use of confocal

microscopy.

Experimental procedure

1. Obtain Arabidopsis seedlings (supplied by the TAs) that represent

wildtype and a selection of transgenic GFP-gene fusion marker lines (Table

1).

Table 1. Transgenic Arabidopsis GFP-fusion marker lines

Plant Line GFP-fused gene present

Ler-0 none present

pEGAD GFP1 cytoplasmic GFP

LTi6b1 SIMIP

ER1 Q4

GFP::TALIN2 Mouse TALIN

GFP::MAP43 MAP4

1. Cutler et al (2000), 2. Kost et al (1998), 3. Marc et al (1998)

2. Using 4-day wild-type seedlings, mount individuals in a drop of FM4-64

dye (4 M solution) on a microscope slide. Stain for 5 minutes, coverslip and

image using the confocal microscope, as demonstrated by your TA. You will

focus on cells in the root apex and hypocotyls of seedlings.

3. Obtain a Z-stack for a wild-type root specimen. When imaging, take note

of parameters such as the laser used, excitation and emission wavelengths

of light source, objective (including magnification) and optical zoom. Note

the number of optical sections used and the distance at which sections are

- 15 -


made. Were any technical difficulties experienced? Using a midsection

image obtained, observe the organization of cell types within the root apex.

Make an estimate of cell sizes within the meristematic region of the root.

4. With each marker line provided, make identical preparations for GFP

imaging. Your TA will help you to visualize cells at higher magnifications.

Note down any changes in parameters used for imaging GFP signal, as

compared to FM4-64. Obtain images of cells that clearly demonstrate GFP

signal for that marker line. Note the appearance of the GFP localization and

whether it corresponds to what is expected.

Questions to consider

1. What is the purpose of using FM4-64 dye in this experiment? What

component of the cell does it stain and how does it help cellular

visualization?

2. What is GFP and from where is this marker protein derived?

3. Describe the individual gene constructs represented in this experiment.

What is known about the genes that are fused to the GFP reporter

protein? Why are these valuable constructs to have?

4. Summarise, with pictures to illustrate, the GFP localization for each

transgenic line.

- 16 -


References

General references on plant structure, development and microtechnique

Esau K 1977 Anatomy of Seed Plants. 2nd ed. Wiley

Jurzitza, G 1987 Anatomie der Samenpflanzen. Thieme Verlag

Raven PH, RF Evert, SE Eichhorn 1999 Biology of Plants. 6th ed. Worth.

Ruzin SE 1999 Plant microtechnique and microscopy. Oxford.

Steeves TA, IJ Sussex 1989 Patterns in plant development. 2nd ed. Cambridge.

Strasburger, E. 2002 Lehrbuch der Botanik für Hochschulen. 35nd ed. Heidelberg

Selected references on wildtype Arabidopsis structure and development

Bowman J (ed.) 1994 Arabidopsis: An atlas of morphology and development. Springer-

Verlag.

Busse JS and RF Evert 1999 Vascular differentiation and transition in the seedling of

Arabidopsis thaliana (Brassicaceae) Int. J. Plant Sci. 160: 241-251.

Dolan LK Janmaat, V Willemsen, P Linstead, S Poethig, K Roberts and B Scheres

1993 Cellular organization of the Arabidopsis thaliana root. Development 119: 71-84.

Pyke KA and RM Leech 1992 Temporal and spatial development of the cells of the

expanding first leaf of Arabidopsis thaliana (L.) Heyng. J. Exp. Bot. 42: 1407-1416.

Schultz EA and GW Haughn 1991 LEAFY, a homeotic gene that regulates

inflorescence development in Arabidopsis. Plant Cell 3: 771-781.

Zhong, R, Taylor JJ, Y ZH 1997 Disruption of interfasicular fiber differentiation in an

Arabidopsis mutant. Plant Cell 9: 2159-2170.

- 17 -


References on cell proliferation and development in Arabidopsis

DeBlock M and D Debrouwer 1992 In situ enzyme histochemistry on plastic-embedded

plant material. The development of an artefact-free -glucuronidase assay. Plant J 2:

261-266.

Donnelly PM, D Bonetta, H Tsukaya, RE Dengler and NG Dengler 1999 Cell cycling

and cell enlargement in developing leaves of Arabidopsis. Devel. Biol. 215: 407-419.

Doonan J 2001 Social controls on cell proliferation in plants. Curr. Op. Plant Biol.

3:482-487.

Doonan J and P Fobert 1997 Conserved and novel regulators of the plant cell cycle.

Curr. Op. in Cell Biol. 9: 824-830.

Ferreira PCG, AS Hemerly, J de Almeida Engler, M Van Montagu, G. Engler and D

Inze 1994 Developmental expression of the Arabidopsis cyclin gene cyc1At. Plant Cell

6: 1763-1774.

Hemerly A, J de Almeida Engler, C Bergounioux, M Van Montagu, G Engler, D Inze

and P Ferreira 1995 Dominant negative mutants of the Cdc2 kinase gene uncouple cell

division from iterative plant development EMBO J 14: 3925-3936.

Hemerly AS, PCG Ferreira M Van Montagu and D Inze 1999 Cell cycle control and

plant morphogenesis: is there an essential link? BioEssays 21: 29-37.

References on cell imaging in Arabidopsis

Cutler SR, Ehrhardt DW, Griffitts JS, and Somerville CR. (2000) Random GFP::cDNA

fusions enable visualization of subcellular structures in cells of Arabidopsis at a high

frequency.

Dolan L, Janmaat K, Willemsen V, Linstead P, Poethig S, Roberts K and Scheres B.

(1993) Cellular organization of the Arabidipsis thaliana root. Development. 119: 71-84.

Kost B, Spielhofer, P and Chua NH. (1998) A GFP-mouse talin fusion protein labels

plant actin filaments in vivo and visualizes the actin cytoskeleton in growing pollen

tubes. Plant J. 16: 393-401.

- 18 -


Marc J, Granger CL, Brincat J, Fisher DD, Kao T, McCubbin, AG and Cyr RJ. (1998) A

GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living

epidermal cells. Plant Cell 10: 1927-1939.

Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G. & Cormier, M. J.

(1992) Primary structure of the Aequorea victoria green fluorescent protein. Gene

111:229233.

Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. & Prasher, D. C. (1994) Green

fluorescent protein as a marker for gene expression. Science 263:802805.

Haseloff, J., Siemering, K., Prasher, D. & Hodge, S. (1995) Stable expression of GFP

in Arabidopsis. Posting on the bionet.genome.arabidopsis newsgroup, 13th June 1995.

Sheen J, Hwang S, Niwa Y, Kobayashi H and Galbraith DW. (1995) Green-fluorescent

protein as a new vital marker in plant cells. Plant J. 8: 777-784.

Websites of interest

Olympus http://www.olympus.de/microscopy/

Ehrhardt Lab http://deepgreen.stanford.edu/

Haseloff Lab http://www.plantsci.cam.ac.uk/Haseloff/Home.html

History of GFP http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP2.htm

- 19 -

http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP2.htm

http://www.plantsci.cam.ac.uk/Haseloff/Home.html

http://deepgreen.stanford.edu/

http://www.olympus.de/microscopy/



Lab 3: Clonal Analysis and Fate

Mapping

- 20 -


Introduction

This laboratory will introduce you to the concept and technique of clonal

analysis and fate mapping.

The developmental history of any structure or organ can be traced back through

its cell lineages to a founding cell or cell population. It is important to be able to

determine the size and identity of the founding cell population in order to

understand the influences that shape the development of a structure or organ.

To follow the cell lineage, cells needs to be marked. The marker needs to be

cell-autonomous and be neutral in a sense that it does not interfer with the

behaviour and development of the marked cell(s). To mark single cells at a

particular time in development, one can for example irradiate a plant, generating

mutations or chromosome breaks at low frequency.The mutations or

chromosomes breaks inactivate or remove the marker gene. For example, the

loss of both active copies of a pigmentation gene can result in a colorless sector

that stands out in the surrounding pigmented tissue. A common approach has

been to delete a pigmentation gene, which is present in heterozygous form and

located near the tip of a chromosome.

Generation of marked cells:

- 21 -


Cell lineages can also be marked by spontaneous events such as the excision

of a transposable element or transposon. One can use reporter gene constructs

such as the uidA gene encoding beta-glucoronidase (GUS) linked through an

Ac transposable element to the CaMV35S promoter (35S::Ac::GUS). The

expression of the GUS reporter gene from this construct is dependent on the

excision of the Ac transposable element. GUS expression can be detected

histologically by a chromogenic substrate that deposits a colored precipitate at

the site of GUS activity. The advantage of using a transposable element is that

cells can be marked at different times during development, if the excision

process is not developmentally regulated.

If the cells are marked early in development and are destined to give rise to

many progeny cells, then the resulting sectors will be large. If cells are marked

late in development fewer divisions will occur after the induction of the clone

and the resulting sectors will be small.

Severals methods for determining the number of founder cells exist: the

determination of the apparent cell number or ACN and sector boundary

analysis.

- 22 -


The apparent cell number concept (ACN)

The principle of sector boundary analysis

- 23 -


During this lab, we will use a 35S::Ac::GUS construct and perform a sector

boundary analysis. The TAs will provide you with Arabidopsis plants at the

rosette stage, which were already stained for GUS activity. You will screen the

plants for sectors, determine their frequency and deduce the number of cells

giving rise to a leave.

Questions to consider

a. what requirements must be met by the optimal clone marker?

b. what are the principles of a sector boundary analysis?

c. how many organ founder cells will eventually make up a leaf?

d. what organisation display the organ founder cells of the leaf?

e. is cell lineage important for leaf development? Why?

f. what is a “structural template”?

References

General references on clonal analysis and fate mapping

Christianson M. L. (1986). Fate map of the organizing shoot apex in Gossypium. Am. J.

Bot. 73, 947-958.

Dawe, R. K. and Freeling, M. (1992). The role of initial cells in maize anther

morphogenesis. Development 116, 1077-1085.

Poethig, R. S. et. al. (1986). Cell lineage patterns in maize Zea mays embryogenesis: a

clonal analysis. Dev. Biol. 117, 392-404.

Poethig, R. S. (1987). Clonal analysis of cell lineage patterns in plant development.

Am. J. Bot. 74, 581-594.

- 24 -


Poethig, R. S. (1989). Genetic mosaics and cell lineage analysis in plants. Trends

Genet 5, 273-277.

Selected references on fate mapping of Arabidopsis organs

Bossinger, G. and Smyth, D. R. (1996). Initiation patterns of flower and floral organ

development in Arabidopsis thaliana. Development 122, 1093-1102.

Jenik, P. D. and Irish, V. F. (2000). Regulation of cell proliferation patterns by homeotic

genes during Arabidopsis floral development. Development 127, 1267-76.

Schnittger et. al. (1996). Epidermal fate map of the Arabidopsis shoot meristem. Dev

Biol 175, 248-55.

- 25 -



Lab 4: Genetic Analysis of Flower

Development

- 26 -


Introduction

In the first part of the practical course will provide you with an opportunity to

derive a genetic model by observation of floral mutant phenotypes and

comparing them with wild-type flowers. Several Arabidopsis mutants defective

in the specification of floral organ identity will be available for you to analyze.

With these, it is possible to construct a simple model that accounts for the

observed phenotypes. The genetic control of flower organ development in

Arabidopsis and Antirrhinum (snapdragon) has been studied extensively, and

the "ABC model" has been derived to explain the mutant phenotypes

(Carpenter and Coen, 1990; Schwarz-Sommer et al., 1990; Bowman et al.,

1991; reviewed in Coen and Meyerowitz, 1991; Weigel and Meyerowitz, 1994).

You will analyze the phenotypes and derive the model for yourself.

In the second part of this lab course you will further test the ABC model.

To explain the phentoypes and derive the ABC model you have to make

assumptions concerning the spatial expression pattern of the various genes.

Using transgenic Arabidopsis plants carrying different promoter::GUS gene

fusion constructs you will test if the various expression data fit the model or not.

Analysis of wild-type and mutant flowers

1. Four flower development mutants (labeled 1, 2, 3 and 4) and wild type

are provided.

- 27 -


2. Try to define how the wild-type Arabidopsis flower looks like: By using

the following diagram, find out which flower organs can be found in which

whorl.

3. Carfully observe the mutants flowers. How many whorls do mutant

flowers have? Indicate which floral whorls are affected in the mutants:

Are flower organs within the whorls absent? Or have they been

converted into other organs?

4. Indicate the domains in which each gene is required in wild type flowers.

(Assume mutant 1 has a null mutation in gene 1; mutant 2 in gene 2; and

mutant 3 in gene 3).

In wild-type flowers, indicate which genes are active in:

Whorl 1: ________________ = Sepals

Whorl 2: ________________ = Petals

Whorl 3: ________________ = Stamens

Whorl 4: ________________ = Carpels

Can this model account for the unique identity of each whorl?

5. Using your model, reconsider the mutants. Indicate the genes that are

most likely active in each whorl in the mutant flowers: (e.g. in mutant 1,

gene 1 function is entirely absent, so only gene 2 and gene 3 activities

are relevant.)

- 28 -

21 3 42.1.

3.

4.


Mutant X: Genes Organs

Whorl 1: ________________ =_______________

Whorl 2: ________________ =_______________

Whorl 3: ________________ =_______________

Whorl 4: ________________ =_______________

6. In the absence of gene 1, is the domain of gene 2 function altered?

Consider other pairwise combinations, and note any alterations from the

wild-type sites of gene activity.

Is it possible that genes 1, 2 and/or 3 regulate each other? If so, how?

Expression analysis of promoter::GUS fusion constructs

Four reporter lines will be handed to you by the TAs. Analyse the blue stain

patterns carefully. Where do you find GUS expression. Do you find it also in

unexpected areas of the flower? Do you think you look at the right stage of floral

development? Do all tissues exhibit staining with the same intensity?

Additional questions to consider:

a. what can reporter genes tell you about gene expression?

b. do you know of other reporter genes? Why do we use GUS?

c. what are the advantages and disadvantages, respectively, of GUS?

c. should the GUS expression correlate with the mutant phenotypes you

observe?

References

Alvarez, J. and Smyth, D. R. (1999). CRABS CLAW and SPATULA, two Arabidopsis

genes that control carpel development in parallel with AGAMOUS. Development 126,

2377-2386.

- 29 -


Bowman, J. L. et.al. (1991). Genetic interactions among floral homeotic genes of

Arabidopsis. Development 112, 1-20.

Bowman, J. L. et. al. (1993). Control of flower development in Arabidopsis thaliana by

APETALA1 and interacting Genes. Development 119, 721-743.

Carpenter, R. and Coen, E. S. (1990). Floral homeotic mutations produced by

transposon-mutagenesis in Antirrhinum majus. Genes Dev. 4, 1483-1493.

Coen, E. S. and Meyerowitz, E. M. (1991). The war of the whorls: genetic interactions

controlling flower development. Nature 353, 31-37.

Krizek, B. A. and Meyerowitz, E. M. (1996). The Arabidopsis homeotic genes

APETALA3 and PISTILLATA are sufficient to provide the B class organ identity

function. Development 122, 11-22.

Schwarz-Sommer, Z., et.al. (1990). Genetic control of flower development: homeotic

genes of Antirrhinum majus. Science 250, 931-936.

Weigel, D. and Meyerowitz, E. M. (1994). The ABCs of floral homeotic genes. Cell, 78,

203-209.

- 30 -



Lab 5: Activation Tagging and Gene

Isolation

- 31 -


INTRODUCTION

(Taken from Weigel et. al. 2000 Plant Physiology 122, pp. 1003-1013)

The primary tool for dissecting a genetic pathway is the screen for loss-of-

function mutations that disrupt such a pathway. However, a limitation of loss-of-

function screens that they rarely identify genes that act redundantly. The

problem of functional redundancy has become particularly apparent during the

past few years, as sequencing of eukaryotic genomes has revealed the

existence of many duplicated genes that are very similar both in their coding

regions and their non-coding, regulatory regions. A second class of genes

whose entire function is difficult to identify with conventional mutagens, which

primarily induce loss-of- function mutations, are those that are required during

multiple stages of the life cycle and whose loss of function results in early

embryonic or in gametophytic lethality. Genes that are not absolutely required

for a certain pathway can still be identified through mutant alleles, if such genes

are sufficient to activate that pathway. Similarly, genes that are essential for

early survival might be identified through mutant alleles if ectopic activation of

the pathways they regulate is compatible with survival of the organism. The key

in either case is the availability of gain-of- function mutations. An example of the

first case is the ethylene response pathway in Arabidopsis. While dominant,

gain-of-function mutations in any of several His kinase genes result in

constitutive repression of the ethylene response, loss-of-function mutations in

individual genes cause no apparent phenotype. However, the combination of

multiple loss-of-function mutations leads to progressive activation of constitutive

ethylene response (Hua and Meyerowitz, 1998). An example of the second

case is provided by the Drosophila homeotic gene Antennapedia (Antp), whose

normal function is to promote the formation of thoracic segments and whose

inactivation results in embryonic lethality (Denell et al., 1981). However, Antp

was originally identified through gain-of-function mutations associated with the

transformation of antenna into leg in the adult fly due expression of a normal

protein product (Gehring, 1967).

- 32 -


Gain-of-function phenotypes can either be mutations in the coding region that

lead to activation of the resulting protein, as in dominant response mutants

(Chang et al., 1993), or by mutations that alter levels or patterns of gene

expression, as in dominant Antp mutants. The traditional way to induce the

latter type of mutation has been through chromosomal rearrangements or

transposons that bring genes under the control of new promoters or enhancers.

A few years ago, a more directed way to induce such mutations was developed

by Walden and colleagues (Hayashi et.al. 1992), who constructed a T-DNA

vector with four copies of an enhancer element from the constitutively active

promoter of the cauliflower mosaic virus (CaMV) 35S gene (Odell et. al., 1985).

These enhancers can cause transcriptional activation of nearby genes, and,

because activated genes will be associated with a T-DNA insertion, this

approach has become known as activation tagging. The original activation-

tagging vector has been used in tissue culture to identify a His kinase from

Arabidopsis, whose overexpression can bypass the requirement for cytokinin in

the regeneration of shoots (Kakimoto, 1996). A related approach, with a

complete CaMV 35S promoter pointing outward from a transposable Ds

element, has been used to identify dominant mutations at the Arabidopsis loci

ELONGATED HYPOCOTYL (LHY), and SHORT INTERNODES (SHI) (Wilson

et al., 1996; Schaffer et.al. 1998; Fridborg et al., 1999).

This laboratory will introduce you to an activation tagging system in Arabidopsis,

which was developed by Detlef Weigel and Joanne Chory at the Plant Biology

Laboratory, The Salk Institute, San Diego and Martin Yanofsky, Dept. of

Biology, UC San Diego. You will undertake all the major steps that are required

for a basic analysis of the gene in question. Essentially the same steps are

taken in a regular research project. You first screen through a population of

activation tagged mutants. You pick a mutant according to certain criteria. You

will isolate the corresponding gene and analyse its primary structure. Using this

sequence information you will search the literature and public databases to get

some first idea about what the gene product may be doing in the plant at the

molecular level. Finally, you will present your findings to the scientific

community (that’s you and your peers….).

- 33 -


Questions to consider:

a. What are the advantages and disadvantages of activation tagging

mutagenesis versus other mutagenesis approaches?

b. Does the result depend on the used promoter? If so, how and why?

c. What is an enhancer?

d. In what regions of the genome is the T-DNA most likely to insert?

e. Why is I-PCR useful? What other approaches could be used to identify

flanking sequences?

LAB PROCEDURE

Depending on the number of students, people will work as separate /

independent teams of 2, or as individuals, to conduct the following experiments.

Note: Record all your experiments and observations in a bound laboratory

notebook. You will need a record later of all the different experiments and

results so that you will be able to write up your results and/or prepare the power

point presentation.

Experiments:

1. Screen for mutants in the activation tagged population

2. Amplification of the genomic DNA flanking the T-DNA insertion site

3. Sequence analysis of the DNA flanking the T-DNA insertion site,

identification of the tagged gene

- 34 -


Experiment 1: Screen for mutants in the activation tagged population

A population of activation tagged mutants will be provided by the TAs. Screen

through the plants and choose a mutant to work with. Explain why you choose

the mutant, describe the mutant phenotype and take pictures of the mutant

plant.

Questions to consider:

a. What criteria do you use to define a mutant?

b. What criteria do you use to select a mutant?

c. When you see a mutant phenotype do you expect it to be recessive or

dominant? Why? How can you determine this?

d. How many plants are growing in the flats? How many plants show a

mutant phenotype? Estimate the efficiency of the method.

e. Why does cold treatment lead to even germination and why is this

necessary?

Experiment 2: Amplification of the genomic DNA flanking the T-DNA insertion site

4.2.1 DNA extraction

To extract DNA, several protocols and kits exist. Different protocols lead to

different amounts and quality of DNA and extraction times. Depending of the

experiment, one needs different quality and amount of DNA. For example, for

PCR, one only needs a few ng of DNA of regular quality.DNA isolated from

plant material is often contaminated with polysaccharides leading to two

problems:

- the DNA pellets are slimy and difficult to handle

- 35 -


- the presence of polysaccharides can inhibit further enzymatic analysis of the

DNA

Some methods for recovering DNA from plants deal with polysaccharide

removal more effectively than others. Caesium chloride density gradients are

time consuming, expensive and technically demanding. CTAB is a commonly

used method relying on the differential solubilities of polysaccharides and DNA

in cetyltrimethylammonium bromide. Different concentrations of CTAB are

required for different plant species and yields can be low. The Dellaporta

method is a quick extraction relying on high concentrations of potassium ions

and SDS to form insoluble complexes with proteins and polysaccharides.

For our experiment, we need DNA of normal quality. Thus, during this

laboratory, we will use a CTAB-DNA extraction method. High concentrations of

NaCl and Sarcosyl were used to precipitate high levels of polysaccharides

(Murray and Thomphoson, 1980).

The protocol can be divided in several steps:

- breaking the cell wall

- cell lysis

- DNA extraction

- DNA precipitation

- Before you start prepare the microprep-buffer (composition see below)

DNA extraction buffer (EB): 200ml 500ml

100 mM Tris-base 20ml (1M) 50ml

5 mM EDTA 2ml (0,5M) 5ml

0.35 M sorbitol 12,75g 31,88g

(Mr=182,18g/mol)

m=MxcxV

- 36 -


Lysis buffer (LB):200ml 500ml

0.2 M Tris-base (pH 8.0) 40ml (1M) 100ml (1M)

50 mM EDTA (pH 8.0) 20ml (0,5M) 50ml (0,5M)

2 M NaCl 80ml (5M) 200ml (5M)

2% CTAB (w/v) 4g 10g

= hexadecyltrimethylammonium bromide

Microprep buffer:

2,5 parts DNA extraction buffer 10/20ml

2,5 parts lysis buffer 10/20ml

1 part 5% Sarcosyl (=N-Lauroylsarcosine, sodiumsalt) 4/8ml

before use add 0,3g sodium bisulfite/100 ml 91.2mg/182.4mg

4.2.1.1 Breaking the cells walls

- collect 1 small plant leave

- grind the tissue with liquid nitrogen with a pestle in an Eppendorf-tube

until the tissue is reduce to powder

4.2.1.2 Cell lysis

- add 750µl of prewarmed microprep-buffer

- vortex 40-60 s until thoroughly mixed

- Incubate at 65°C for 30-60 min

4.2.1.3 DNA extraction

- add 750 l Chloroform:isoamyl (IAA = Isoamylalkohol or indol acetic acid or

3-Methyl-1-butanol or Isopentylalcohol) (24:1)

- mix well by vortex

- centrifuge for 10 min at 10.000 rpm

- Chloroform removes complex proteins/ polysaccharides

- 37 -


- pipette off the upper DNA containing aqueous phase (approximately 0,5ml) into

a fresh tube.

4.2.1.4 DNA precipitation

- add 2/3 to 1 times the volume of cold isopropanol

- invert tubes repeatedly until DNA precipitates

- immediately spin at 13.000 rpm for 20 min at 4 C

- discard the supernatant

- wash the DNA pellet with cold 70% ethanol

- spin at 13.000 rpm for 10 min

- dry pellet with speed vac 5 min

- resuspend the DNA in 50 l 1xTE (containing 0.2mg/ml RNase)

- incubate at 37C for 1 h

- store DNA at -20C

-4.2.1.5 DNA concentration

- check the DNA concentration by measuring the OD at 260/280 nm

- at 260 nm, 1 OD corresponds to 50 µg/ml of double-stranded DNA.

- the ratio 260/280nm provides an estimate of the purity of the DNA. Pure

DNA have a value of 1.8.

4.2.2 TAIL-PCR

1. PCR-Mix: primary reactiona. 10x buffer 2 lb. MgCl2 (25 mM) 2 lc. dNTPs (2 mM) 2 ld. primer TR1 (2 µM) 2 le. primer AD1 (20 µM) 2 l

or AD2 (30 µM)or AD3 (40 µM)

f. template (20ng DNA) 2 µl g. taq polymerase (1U/µl) 0.3 µl

= 20 l

- keep 3µl for secondary reaction

- 38 -


2. PCR-Mix: secondary reaction

a. 10x buffer 2 lb. MgCl2 (25 mM) 2 lc. dNTPs (2 mM) 2 ld. primer TR2 (2 µM) 2 le. primer AD1 (20 µM) 2 l


f. template (1:50 dilution from primary reaction) 1 µl 3µl from primary reaction + 147 µl H2O

g. taq polymerase (1U/µl) 0.3 µl = 20 l

- keep 3 µl for tertiary reaction

3. PCR-Mix: tertiary PCR reactiona. 10x buffer 10 lb. MgCl2 (25 mM) 10 lc. dNTPs (2 mM) 10 ld. primer TR3 (2 µM) 10 le. primer AD1 (20 µM) 10 l


f. template (1:10 dilution of sec. PCR product; 1 µl 3µl from primary reaction + 27 µl H2O)

g. taq polymerase (1U/µl) 1,5 µl = 100l

The PCR parameters:

The primary PCR: TAIL-A (PCR-program 36), time 5:50h

cycles Parameters

1 93 °C 1min, 95 °C 1 min

5 94 °C 30 sec, 62 °C 1 min, 72 °C 2.5 min

1 94 °C 30 sec, 25 °C 3 min, Ramping to 72 °C over 3 min, 72 °C 2.5 min

20 94 °C 10 sec, 68 °C 1 min, 72 °C 2.5 min; 94 °C 10 sec, 68 °C 1 min, 72 °C

- 39 -


2.5 min; 94 °C 10 sec, 44 °C 1 min, 72 °C 2.5 min

1 72 °C 5min, 4°C ∞

The secondary PCR: TAIL-B (PCR-program 37), time 4:40h

cycles parameters

18 94 °C 10 sec, 64 °C 1 min, 72 °C 2.5 min; 94 °C 10 sec, 64 °C 1 min, 72 °C

2.5 min; 94 °C 10 sec, 44 °C 1 min, 72 °C 2.5 min

1 72 °C 5min, 4°C ∞

The tertiary PCR: TAIL-C (PCR-program 38), time 2:03h

cycles parameters

20 94 °C 15 sec, 44 °C 1 min, 72 °C

2.5 min

1 72 °C 5min, 4°C ∞

4.2.3 Agarose gel

- make a 1% TAE agarose gel

- load the PCR reaction

- run the gel at 100 V for 1h

- check the gel under the UV (take care! Use gloves, covering shields and wear glasses)

- take a picture

4.2.4 Gel extraction

To extract the band (only from the secondary PCR-reaction) from the agarose

gel, we will use a kit from Qiagen called QIAquick® Gel Extraction Kit. The kit

uses a chaotropic agent that denatures protein, dissolves agarose, and

promotes the binding of double-stranded DNA (100 base-pairs to 48 kilobase-

pairs) to a glass fiber matrix. Once the DNA is "captured", proteins and salt

contaminants are washed away, and the purified DNA is eluted in a low ionic

strength buffer (TE, Tris-HCl or H2O). DNA samples are recovered in a

concentrated form by eluting with as little as 10 µl of buffer or water. QIAquick®

- 40 -


Gel Extraction Kit can be used to purify DNA (e.g., PCR products, restriction

fragments) from solution and from both TAE and TBE agarose gel bands.

Typical recoveries are >80% from solution and > 60% from gel bands. Qiagen

purification of PCR products from solution removes 99.5% of primers and free

nucleotides and can be performed in less than 5 minutes. DNA purification from

a gel slices can be completed in just 15 minutes. Purified DNA can be used

directly without an alcohol precipitation in a variety of applications including

PCR amplification, restriction digest analysis and subcloning.

The maximum weight of gel slice that can be processed with the following pro-

tocol is 400 mg.

- weigh an empty 1.5 ml microcentrifuge tube to the nearest 10 mg and

record the weight

- using a clean razor blade or scalpel, excise the slice of agarose

containing the DNA band to be purified. Cut as close to the DNA band as

possible

- cut the slice into several smaller pieces and transfer them to the pre-

weighed 1.5 ml microcentrifuge tube

- weigh the tube containing the agarose slice to the nearest 10 mg, and

subtract the weight of the empty tube to determine the weight of the slice

- to the gel slice add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~

100 ul).

- close the tube and incubate at 50°C until the agarose is completely

dissolved (5-10 minutes). Vortex vigorously on occasion to aid dissolving

process.

- during the incubation, prepare one QIAquick Column for each

purification.

- after the agarose is completely dissolved, add 1 gel volume of

isopropanol to the sample and mix. Do not centrifuge the sample at this

stage.

- transfer the sample to the prepared QIAquick Column. Incubate at room

temperature for 1 minute

- 41 -


- centrifuge in a microcentrifuge at full speed for 30 seconds

- discard the flow-through by emptying the Collection Tube. Place the

QIAquick Column back inside the collection tube

- add 750 µl of Buffer PE to the column. Centrifuge at full speed for 30

seconds.

- discard the flow-through by emptying the Collection Tube and spin for an

additional 1 min at full speed.

- discard the collection tube and transfer the QIAquick Column to a fresh

1.5 ml microcentrifuge tube (i.e. not a collection tube)

- apply 50 µl of dH20 directly to the top of the glass fiber matrix in the

QIAquick Column

- incubate the sample at room temperature for 1 minute

- centrifuge at full speed for 1 minute to recover the purified DNA

The concentration of the DNA should now be estimated. We will use a method

known as “spot test”. The control includes samples of known DNA

concentration.

- dilute the DNA solution 1 to 10 in dH2O

- set up the following reaction:

Table 1 Spot test

PCR fragment Diluted PCR fragment

Control samples

5 ng/l 10 ng/l 15 ng/l 20 ng/l

(1 g/ml) EthBr 5 l 5 l 5 l 5 l 5 l 5 lDNA 1 l 1 l 1 l 1 l 1 l 1 ldH2O 4 l 4 l 4 l 4 l 4 l 4 l

total volume 10 l 10 l 10 l 10 l 10 l 10 l

- spot the different mixes onto a saran wrap overlaying a regular UV

transilluminator and estimate the appropriate DNA concentration by

- 42 -


comparing the fluorescence intensity of the test samples and the control

samples. Same UV precautious measures as detailed above apply!

This technique provides a rapid way to make a rough but useful estimate of the

DNA concentration of a given sample (e.g. isolated fragment). It is not highly

accurate. Take care to make the comparison in the 5-15 ng range. Above 15-20

ng saturation is reached and no decent estimates can be made based on the

fluorescence intensity. Furthermore, less EthBr intercalates in small DNA

fragments (ca 0.5 kb) than in larger DNA fragments (3 kb). Thus, the method

leads to underestimates of the concentration of small DNA fragments. Don't

wait too long when making the comparison to avoid false results due to drying

of the samples. Nevertheless, we regularly use this method for purposes such

as cloning or probe labeling.

4.2.5 Sequencing PCRYour PCR products will be sequenced through a commercial automated

sequencing service, provided by MWG Biotech. For each sequencing reaction,

30 ng per 100 bp of purified PCR product is required, along with sufficient

primer for the reactions (separate). Based on the size of your PCR product, you

will prepare the appropriate amounts of PCR product in separate tubes, to be

sent to MWG.

Previously, manual sequencing was performed using the following protocol.

The amount of DNA used for the sequencing reaction depends on the size of the DNA fragment.

To determine the corresponding values check the following table:

Table 2

Length PCR fragment Amount of DNA

0.1 to 0.2 kb 1 to 3 ng

0.2 to 0.5 kb 3 to 10 ng

0.5 to 1.0 kb 5 to 20 ng

1.0 to 2.0 kb 10 to 40 ng

- 43 -


> 2.0 kb 40 to 100 ng

- set up the following sequencing-reaction

5x buffer 2µl

primer TR2 (1290) 1µl

PCR-product xµl

Big Dye mix 2µl

Total volume 10µl

Use the following program:

Step 1: 96o C 2 min

Step 2: 96o C 10s

Step 3: 51o C 5s

Step 4: 60o C 4 min back to step 2 24x

Step 5: 4o C forever

The PCR products need to be purified to remove the primers:

- transfer the PCR reaction to a 1.5 ml eppendorf tube

- add 5 µl of 25 mM ECTA (pH 8,0) + 60 µl of 100% ethanol

- mix and leave at RT for 15 min

- centrifuge full speed at 4 oC for 20 min

- remove carefully the supernatant; pellet may not be visible

- wash with 60 µl 70% ethanol and spin full speed at 4o C for 10 min

- dry samples 1 min at 90 oC

- give the samples to the TAs for loading on the sequencer

Questions:

a. Why is DNA extraction necessary, couldn’t you do the PCR directly on

the tissue?

b. What is the function of the various chemical constituents of the different

buffers and the isopropanol?

c. What is the mechanism of the sequencing reaction?

- 44 -


d. Are you sure you analyse the correct gene? What pitfalls can you

encounter?

References

Key references for activation tagging:

Li et al. (2001). BRS1, a serine carboxypeptidase, regulates BRI1 signaling in

Arabidopsis thaliana. PNAS 98, 5922-5926.

Li et. al. (2002). BAK1, an Arabidopsis LRR receptor-like protein kinase, interacts with

BRI1 and modulates brassinosteroid signaling. Cell 110, 213-222.

Kardailsky et. al. (1999). Activation tagging of the floral inducer FT. Science 286, 1962-

1965.

van der Graaff et. al. (2002). Activation tagging of the two closely linked genes LEP

and VAS independently affects vascular cell number. Plant Journal 32, 819-830.

Weigel et. al. (2000). Activation tagging in Arabidopsis. Plant Physiology 122, 1003-

1013.

Additional references:

Chang et. al. (1993). Arabidopsis ethylene-response gene ETR1: similarity of product

to two-component regulators. Science 262, 539-544.

Denell et. al. (1981). Developmental studies of lethality associated with the

Antennapedia gene complex in Drosophila melanogaster. Dev Biol 81, 43-50.

Fridborg et. al. (1999). The Arabidopsis dwarf mutant shi exhibits reduced gibberellin

responses conferred by overexpression of a new putative zinc finger portein. Plant Cell

11, 1019-1032.

- 45 -


Gehring W (1967). Bildung eines vollständigen Mittelbeins mit Sternopleura in der

Antennenregion bei der Mutante Nasobemia (Ns) von Drososophila melanogaster.

Arch Julius Klaus Stift Vererbungsforsch Sozialanthropol Rassenhyg 41, 44-54.

Hayashi et. al. (1992). Activation of a plant gene by T-DNA tagging: auxin-independent

growth in vitro. Science 258, 1350-1353.

Hua J and Meyerowitz EM (1998). Ethylene responses are negatively regulated by a

receptor gene family in Arabidopsis thaliana. Cell 94, 261-271.

Kakimoto T (1996). CKI1, a histidine kinase homolog implicated in cytokinin signal

transduction. Science 274, 982-985.

Odell et. al. (1985). Identification of DNA-sequences required for activity of the

cauliflower mosaic virus-25S promoter. Nature 313, 810-812.

Schaffer et. al. (1998). The late elongated hypocotyl mutation of Arabidopsis disrupts

circadian rhythms and the photoperiodic control of flowering. Cell 93, 1219-1229.

Wilson et. al. (1996). A Dissociation insertion causes a semidominant mutation that

increases expression of TINY, an Arabidopsis gene related to APETALA2. Plant Cell 8,

659-671.

Liu, Y. G. and R. F. Whittier (1995). "Thermal asymmetric interlaced PCR: automatable

amplification and sequencing of insert end fragments from P1 and YAC clones for

chromosome walking." Genomics

Liu, Y. G., N. Mitsukawa, et al. (1995). "Efficient isolation and mapping of Arabidopsis

thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR." Plant J

Experiment 3. Sequence analysis of the DNA flanking the T-DNA insertion site and identification of the tagged gene.

Bioinformatics is an essential component of molecular genetics. The following

website provides you with 'The ABC of Bioinformatics':

“http://acer.gen.tcd.ie/embarc/abccont.html”.

- 46 -


You will use the DNA nucleotide sequences you have identified to run BLAST

searches to identify similar sequences in the databases. Blast finds sequences

in a database that are similar to a query sequence. It uses the method of

Altschul et al. (1990). The query sequence and the database to be searched

can be either peptide or nucleic acid (NA) sequence in any combination.

Basic BLAST tools:

blastp: compares a protein sequence against a protein DB

blastn: compares a NA sequence against a NA DB

blastx: dynamically translates a NA query in all 6 reading frames and

compares it to a protein DB

tblastn: compares a protein query against a NA database translated in all

six reading frames

tblastx: translates both input NA and NA database 6x and compares

As the Arabidopsis genome has now been fully sequenced you can identify

where in the Arabidopsis genome your T-DNA insertion is located by performing

a BLAST search against the Arabidopsis genome sequence. For this go to the

following URL: “http://www.arabidopsis.org/Blast/”.

Below is the first part of the output from a search for the yeast homologue of the

prokaryotic recA gene, using recA from E.coli as the query sequence.

Output from http://www.ncbi.nlm.nih.gov/BLAST

Score E

- 47 -


Sequences producing significant alignments: (bits)

Value

gi|603333 (U18839) Rad51p: RecA-like protein [Saccharomyces cer.] 52 1e-07

gi|603420 (U18922) Dmc1p: DNA repair protein [Saccharomyces cer.] 40 8e-04

gi|642809 (Z48008) Rad57p [Saccharomyces cerevisiae] 34 0.061

gnl|PID|e252186 (Z75270) ORF YOR362c [Saccharomyces cerevisiae] 32 0.14

gi|500681 (U10398) Yhr131cp [Saccharomyces cerevisiae] 28 2.7

The hits are reported as gi numbers - a unique identifier for the Genbank

sequence, followed by a Genbank/EMBL accession number, then a brief

descriptor, then a score in bits (which should be independent of the scoring

matrix) and finally an E-value. The latter is the same as the -exp parameter and

tells you the number of hits you would expect in the searched database by

chance alone. 1e-07 is a small number so the match is likely to be statistically

(and biologically!) significant. E-values of 0.1 or 0.05 are usually used as cutoffs

in sequence database searches. Using a larger E-value cutoff in a database

search allows more distant matches to be found, but also results in a higher rate

of spurious alignments. A large E-value (for example 5 or 10) indicates that the

alignment is most likely due to chance as this similarity has a chance of 5 out of

100 (1 in 20) to occur by chance alone. This doesn’t sound so bad but is

unlikely to be biologically meaningful. Further analysis has to be done. There is

also a limit, about 25% sequence similarity for proteins of normal length, beyond

which sequence similarity becomes uninformative. If you encounter such a low

(or lower) level of relatedness you need additional information, for example from

structural analyses, to back up your hypothesis.

1. Ensure that your sequence contains no sequence from the T-DNA element

before submitting it to the BLAST search (i.e. trim the sequence)

2. Perform a BLAST search and identify where on the genome the T-DNA is

inserted.

- 48 -


3. For genes identified conduct a literature search to determine whether

mutants have already been described in these genes and what the likely

function of the genes are.

- 49 -


Questions:

a. If two genes exhibit high levels of sequence similarity does this mean

they are homologous? How can you demonstrate homology?

b. If your T-DNA is inserted in a region where there is no gene or coding

region predicted in the Arabidopsis genome does this mean that it will

have no phenotype?

c. When would you use the amino-acid sequence of a protein to search for

similar genes rather than the underlying nucleic acid sequence?

d. If you did not have access to a fully sequenced Arabidopsis genome how

would you identify the location and nature of the T-DNA tagged region?

e. How do you deal with the gaps in the observed alignments in homology

searches?

References

Altschul et. al. (1990). Basic local alignment search tool. J. Mol. Biol. 215, 403-410.

Gibas, C. and Jambeck, P. (2001). Developing bioinformatics computer skills. O’Reilly

& Associates, Inc, Sebastopol, CA, USA.

Lesk, A. M. (2002). Bioinformatik. Eine Einführung. Spektrum Akademischer Verlag,

Heidelberg.

NCBI home page: “http://www.ncbi.nlm.nih.gov/”.

NCBI Handbook: http://www.ncbi.nlm.nih.gov/books/bv.fcgi?

call=bv.View..ShowTOC&rid=handbook.TOC&depth=2.

McLysaght, A. and Lloyd A. The ABC of bioinformatics.

http://acer.gen.tcd.ie/embarc/abccont.html.

The protein information resource: http://pir.georgetown.edu/pirwww/pirhome3.shtml.

- 50 -



Lab 6: Analysis of Gene, Literature Search and Presentation of

Results

- 51 -


Introduction

In this lab you get acquainted with analysing your gene in more detail using

web-based sequence analysis tools, doing a literature search, extracting the

necessary information regarding your gene from the various sources of

information, and presenting your data to your peers. Probably you have already

realised that nowadays information is not just extracted from papers published

in a scientifc journal. Still, the journals are the most important source of

information and moste of them have a website where one can browse or

download articles. Useful information, however, can also be obtained from other

places such as publically accessible databases including GenBank, PDB and

other resources accessible via the internet.

Sequence analysis of geneOnce you have identified your gene you want to learn as much as possible

about the structure of the putative protein, what domains constitute your protein,

what does it do biochemically, where is it located in the cell etc. Often you can

get good preliminary information about those characteristics of your gene using

a set of sequence analysis tools (see below). Note that those tools give you

indications only. You still have to do experiments to actually show that some

suggested properties for the protein, such as the compartimental localisation or

wether it possibly acts as a certain type of transcription factor, are correct.

- 52 -


An incomplete list of commonly used websites for sequence analysis.

http://www.yorvic.york.ac.uk/ppc/sequence.html#homology%20search

http://www.ncbi.nlm.nih.gov/

http://pir.georgetown.edu/pirwww/pirhome3.shtml

http://smart.embl-heidelberg.de/

http://www.ebi.ac.uk/embl/index.html

http://www.expasy.org/

http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml

http://www.arabidopsis.org/tools/

http://mips.gsf.de/proj/thal/db/main.html

Literature searchA convenient starting point in a literature search is PubMed, the literature

database run by the NCBI in the US. The following introduction to PubMed is

copied from the NCBI Handbook Chapter 2; PubMed: The Biobliographic

Database by Kathi Canese, Jennifer Jentsch and Carol Myers. Another starting

point is the “Web of Science” website from Thompson/ISI. It is available for

TUM members (see proxy setting issue below). It is a bit less user friendly than

PubMed but at least in the past covered more plant journals. These days,

however, PubMed as a decent coverage of plant journals. In either case you

start your search by typing in author names or key words (e.g., ovule, pattern

formation, homeotic gene) to start a search. Analysing the search results is a

cumbersome and time consuming issue and sometimes many entries have to

be evaluated manually.

Eventually you have to access papers published in individual scientific journals.

Recent paper issues of leading journals can be found in the library. However,

these days it is most convenient to access the journals online via the WWW. To

access a lot of different scientific journals online use the electronic library of the

TUM (EZB) website as a starting portal. The EZB allows access to hundreds of

journals free of charge for members and students of the TUM.

- 53 -


This method, however, works only when connecting to the internet through the

LRZ München and when the proxy settings of your browser are correct.

For EZB-TUM to work please make sure that your proxy settings in the browser are correct! In Netscape/Mozilla Preferences Advanced

Proxies: activate “automatic proxy configuration URL” and type in the following

URL: http://pac.lrz-muenchen.de. IE Mac OS X does NOT work. In IE PC

Tools Internet Options Connections LAN Settings check “use

automatic configuration script”, type the following URL in the address box:

“http://pac.lrz-muenchen.de”.

Once you saved the URL of the journal’s website in your bookmarks you don’t

have to go via the EZB home page to the journal’s website (but keep your proxy

settings). Articles can be read directly on the screen or can be downloaded as

PDF files and printed out. In more recently published issues, usually starting

around 1998, the figures can be downloaded and used in your PPT

presentation. Note, however, that depending on the journal important copyright

issues exist and that figures can be downloaded for personal use only.

Using PubMedSimple SearchingA simple search can be conducted from the PubMed homepage by entering

terms in the query box and pressing Enter from the keyboard or the Go button

on the screen. If more than one term is entered in the query box, PubMed will

go through the Automatic Term Mapping protocol described in the previous

section, first looking for all the terms, as typed, to find an exact match. If the

exact phrase is not found, PubMed clips a term off the end and repeats

Automatic Term Mapping, again looking for an exact match, but this time to the

abbreviated query. This continues until none of the words are found in any one

of the translation tables. In this case, PubMed combines terms (with the AND

Boolean operator) and applies the Automatic Term Mapping process to each

individual word. PubMed ignores Stopwords, such as “about”, “of”, or “what”.

- 54 -


People can also apply their own Boolean operators (AND, OR, NOT) to multiple

search terms; the Boolean operators must be in uppercase.

Search term: vitamin c common coldTranslated as: ((“ascorbic acid” [MeSH

Terms] OR vitamin c [Text Word]) AND (“common cold” [MeSH Terms] OR

common cold [Text Word]))Search term: single cell separation brainTranslated

as: (((“single person” [MeSH Terms] OR single [Text Word]) AND (“cell

separation” [MeSH Terms] OR cell separation [Text Word])) AND (“brain”

[MeSH Terms] OR brain [Text Word])) If a phrase of more than two terms is not

found in any translation table, then the last word of the phrase is dropped, and

the remainder of the phrase is sent through the entire process again. This

continues, removing one word at a time, until a match is found. If there is no

match found in the phrase dictionary or in the Automatic Term Mapping

process, the individual terms will be combined with AND and searched in All

Fields. One can see how PubMed interpreted a search by selecting Details from

the Features Bar on the PubMed search pages after completing a search. For

more information, see Details.

Complex SearchingThere are a variety of ways that PubMed can be searched in a more

sophisticated manner than simply typing search terms into the search box and

selecting Go. It is possible to construct complex search strategies using

Boolean operators and the various functions listed below, provided in the

Features Bar:

• Limits [http://www.ncbi.nlm.nih.gov/entrez/query/static/help/pmhelp.

html#Limits] restricts search terms to a specific search field.

• Preview/Index [http://www.ncbi.nlm.nih.gov/entrez/query/static/help/pmhelp.

html#Index] allows users to view and select terms from search field indexes and

to preview the number of search results before displaying citations.

- 55 -


• History [http://www.ncbi.nlm.nih.gov/entrez/query/static/help/pmhelp.

html#History] holds previous search strategies and results. The results can be

combined to make new searches.

• Clipboard [http://www.ncbi.nlm.nih.gov/entrez/query/static/help/pmhelp.

html#Clipboard] allows users to save or view selected citations from one search

or several searches.

• Details [http://www.ncbi.nlm.nih.gov/entrez/query/static/help/pmhelp.

html#Details] displays the search strategy as it was translated by PubMed,

including error messages.

Additional PubMed FeaturesThe following resources are available to facilitate effective searches:

• MeSH Browser [http://www.ncbi.nlm .nih.gov/entrez/query/static/help/pmhelp.

html#MeSHBrowser] allows searching of MeSH, NLM's controlled vocabulary.

Users can find MeSH terms appropriate to a search strategy, obtain information

about each term, and view the terms within their hierarchical structure.

• Clinical Queries [http://www.ncbi.nlm .nih.gov/entrez/query/static/help/pmhelp.

html#ClinicalQueries ] is a set of search filters developed for clinicians to

retrieve clinical st udies of the etiology, prognosis, diagnosis, prevention, or

treatment of disorders. The Systematic Reviews feature retrieves systematic

reviews and metaanal ysis studies by topic.

• Journal Database [http://www.ncbi.nlm

.nih.gov/entrez/query/static/help/pmhelp. html#JournalBrowser] allows searches

of journal names, MEDLINE abbreviations, or ISSN numbers for journals that

are included in the Entrez system. A list of journals with links to full text is also

included.

• Single Citation Matcher [http://www.ncbi.nlm .nih.gov/entrez/query/static/help/

pmhelp.html#SingleCi tationMatcher] is a “fill-in-the-blank” form that allows a

user to find the Pub Med ID (PMID) number for a single article or all citations in

a given journal i ssue by entering partial journal citation information.

• Batch Citation Matcher [http://www.ncbi.nlm.nih.gov/entrez/query/static/help/

pmhelp.html#BatchCitationMatcher] allows users to find PMID numbers that

correspond to their own list of citations. Publishers or other database providers

- 56 -


who want to link directly from bibliographic references on their websites to

entries in PubMed use this service frequently.

• Cubby [http://www.ncbi.nlm .nih.gov/entrez/query/static/help/pmhelp.

html#Cubby] is a place for users to store search strategies, LinkOut

preferences, and changes to the default Document Delivery Services

[http://www.ncbi.nlm.nih. gov/entrez/query/sta

tic/help/pmhelp.html#DocumentDeliveryServices].

ResultsPubMed retrieves and displays search results in the Summary format in the

order the record was initially added to PubMed, with the most recent first. (Note

that this date can differ widely from the publication date.) Citations can be

viewed in several other formats and can be sorted, saved, and printed, or the

full text can be ordered.

Links from PubMedA variety of links can be found on PubMed citations including:

Related Articles, which retrieves a precalculated set of PubMed citations

that are closely related to the selected article. PubMed creates this set by

comparing words from the title, abstract, and MeSH terms using a word-

weighted algorithm.

LinkOut, which provides links to publishers, aggregators, libraries,

biological databases, sequencing centers, and other websites. These link to the

provider's site to obtain the full text of articles or related resources, e.g.,

consumer health information or molecular biology database records. There may

be a charge to access the text or information, depending on the policy of the

provider.

Books, which provides links to textbooks so that users can explore

unfamiliar concepts found in search results. In collaboration with book

publishers, NCBI is adapting textbooks for the web and linking them to PubMed.

The Books link displays a facsimile of the abstract, in which some words or

phrases show up as hypertext links to the corresponding terms in the books

- 57 -


available at NCBI. Selecting a hyperlinked word or phrase takes you to a list of

book entries in which the phrase is found.

Entrez database, which links to other resources, or NCBI databases may

be available from the links to the right of each citation and from the Display pull-

down menu. PubMed will return only the first 500 items when using the Display

pull-down menu, from which the following links are available:

• Protein [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Protein] – amino

acid

(protein) sequences from SWISS-PROT, PIR, PRF, and PDB and translated

protein sequences from the DNA sequences databases.

• Nucleotide [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide] –

DNA sequences from GenBank, EMBL, and DDBJ.

• PopSet [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Popset] – aligned

sequences submitted as a set from a population, phylogenetic, or mutation

study describing such events as evolution and population variation.

• Structure [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Structure] –

threedimensional structures from the Molecular Modeling Database (MMDB)

that were determined by X-ray crystallography and NMR spectroscopy.

• Genome [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Genome] –

records and graphic displays of entire genomes and chromosomes for

megabase-scale sequences.

• ProbeSet [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=geo] – gene

expression data repository and online resource for the retrieval of gene

expression data from any organism or artificial source.

• OMIM [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM] – directory of

human genes and genetic disorders.

• SNP [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=SNP] – dbSNP is a

database of single nucleotide polymorphisms.

• Domains [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=DOMAINS] – The

Domains database is used to identify the conserved domains present in a

protein sequence.

- 58 -


Questions

a. what is the basic molecular nature of my protein (e.g., transcription

factor, receptor-like kinase)? What domains make up the protein? What

is the function of the individual domains? What is the prediction about

intracellular localisation of the protein? How could you confirm the

prediction?

b. what is the biological function of the gene? Can I explain the mutant

phenotype based on the molecular nature of the predicted protein?

c. what does the literature say about this gene/protein? What does the

literature say about homologous proteins in Arabidopsis or in other

species?

d. what is the best way to design a slide? How much information can I put

on one slide? Can everybody read the text on my slide? Are my slides

OK for color-impaired people? Is my background too dark, too light, too

“nervous”? Do I focus on the data or do I have too many distracting

things on the slide? Am I misleading people with the presentation of the

data?

References

NCBI PubMed home page: “http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?

db=PubMed”.

NCBI Handbook: http://www.ncbi.nlm.nih.gov/books/bv.fcgi?

call=bv.View..ShowTOC&rid=handbook.TOC&depth=2.

The website of the TUM electronic library (Elektronische Zeitschriftenbibliothek, EZB):

“http://www.ub.tum.de/bib/zb_onl.html”.

Web of Science: “http://isi2.isiknowledge.com/portal.cgi” (click on web of science link,

same proxy setting issue applies as for EZB-TUM).

- 59 -


MaterialsGUS Staining buffer

100 mM sodium phosphate pH 7

10 mM EDTA

0.1% Triton X-100

1 mg/ml X-Gluc*

0.5 mM potassium ferricyanide+

0.5 mM potassium ferrocyanide+

* X-Gluc is VERY expensive. Treat stocks with care.

+ Oxidative catalyst. Concentration can be varied from 0-5 mM

Some staining protocols are modified depending on the purposes: a mild fixation before

or after staining of the tissue can be necessary (for instance: ovules and seeds).

Chloramphenicol can be added at 100 µg/mL to inhibits bacterial growth during long

incubation. You may also alter the time of staining; for long staining times (2-3 days) it

is advisable to use 2 - 5 mM ferri/ferrocyanide to limit stain diffusion.

Lactophenol

Lactic acid 25 ml

Glycerol 50 ml

Distilled water 25 ml

Phenol, cryst 25 g

Warm on a water bath until the phenol has gone into solution: then store in a dark glass bottle.

Lactic acid (L-1250, Sigma, 85% (w/w))

Toluidine Blue O0.05% toluidine blue in dest. Water

Sigma (T-3260)

Websites with classical microscopy staining recipies:http://www.hoslink.com/histo/histo_recipes_index.htm

- 60 -


http://www.aeisner.de/index.html

- 61 -

Mittelbaukurse June 18-July 5,...

Documents

Transcript of Mittelbaukurse June 18-July 5,...